Introduction of acetyl-phosphate bypass and increased culture temperatures enhanced growth-coupled poly-hydroxybutyrate production in the marine cyanobacterium Synechococcus sp. PCC7002.

Abstract

Polyhydroxyalkanoate (PHA) is an attractive bio-degradable plastic alternative to petrochemical plastics. Photosynthetic cyanobacteria accumulate biomass by fixing atmospheric CO₂, making them promising hosts for sustainable PHA production. Conventional PHA production in cyanobacteria requires prolonged cultivation under nutrient limitation to accumulate cellular PHA. In this study, we developed a system for growth-coupled production of the PHA poly-hydroxybutyrate (PHB), using the marine cyanobacterium Synechococcus sp. PCC 7002. A recombinant strain termed KB1 expressing a set of heterologous PHB biosynthesis genes (phaA/phaB from Cupriavidus necator H16 and phaE/phaC from Synechocystis sp. PCC 6803) accumulated substantial PHB during growth (11.4% of dry cell weight). To improve PHB accumulation, we introduced the Pseudomonas aeruginosa phosphoketolase gene (pk) into strain KB1, rewiring intermediates of the Calvin-Benson-Bassham (CBB) cycle (xyluose-5-phosphate, sedoheptulose 7-phosphate, and fructose-6-phosphate) to acetyl-CoA. The pk-expressing strain, KB15, accumulated 2.1-fold enhanced levels of PHB (23.8% of dried cell weight), relative to the parent strain, KB1. The highest PHB titer of KB15 strain supplemented with acetate was about 1.1 g L^-1 and the yield was further enhanced by 2.6-fold following growth at 38 °C (0.21 g L^-1 d^-1), relative to growth at 30 °C. Metabolome analysis revealed that pool sizes of CBB intermediates decreased, while levels of acetyl-CoA increased in strain KB15 compared with strain KB1, and this increase was further enhanced following growth at 38 °C. Our data demonstrate that acetyl-phosphate generated by Pk was converted into acetyl-CoA via acetate by hitherto unidentified enzymes. In conclusion, expression of heterologous PHB biosynthesis genes enabled growth-coupled PHB production in strain PCC 7002, which was increased through acetyl-CoA supplementation by bypassing acetyl-phosphate and elevating culture temperature.